X-Ray Crystallography

X-Ray Crystallography is a biophysical method to characterize the structure of proteins by determining the positions of each atom using X-ray diffraction data. This process entails:

  • Crystallization Trials
  • X-Ray Diffraction Data Collection
  • Phase Determination
  • Structural Refinement of Atomic Coordinates
  • Structural Analysis

Crystal of human interleukin-8.

Solved crystal structures may result in three-dimensional atomic coordinates of a biomedically, relevant target protein that can be used for Lead Discovery. The solved structures of lead compounds bound to validated protein targets serve as a valuable basis for Lead Optimization.

Crystallization Trials

Purified proteins are subject to a diverse set of crystallization conditions over a range of precipitants, salts and pH values. Typically crystallization is achieved by the vapor diffusion hanging drop method under one or two sets of conditions. These crystallization conditions can then be optimized to achieve the highest quality crystal growth through adjustment of the precipitant concentration, salts and pH.

Data Collection

Once crystal growth has been achieved, data collection can proceed. Single crystals are mounted and exposed to a high-intensity X-ray beam. X-rays diffract off planes formed by the orderly arrangement of atoms of each protein within the crystal lattice; the periodicity of the crystal amplifies the resulting signal to measurable levels and the signal is recorded by a special detector. The resulting X-ray diffraction data is a set of reflections, or spots, collected in a series of images. Collecting structural information about a novel protein from crystals therefore requires a powerful source of X-rays. The structure-based drug design core is affiliated with one of the world’s most advanced synchrotrons, National Synchrotron Light Source II at the Brookhaven National Laboratory, a Department of Energy-funded facility. In particular, we work with Highly Automated Macromolecular Crystallography Beamline AMX  17-ID-1.

Structure Determination & Refinement

A portion of an electron density map calculated to 1.05 Å (blue) and fitted model of human Interleukin-8. The structure of a disulfide bond (green) is clearly seen in the shape of the map.

Finally, the collected X-ray diffraction data sets must be computationally analyzed and the three-dimensional structure of the protein determined. This is accomplished through the use of a wide variety of computer software packages to calculate the phases of diffracted X-rays, electron density maps and to permit refinement of atomic coordinates. The collection of two-dimensional reflections comprising the X-ray diffraction data set is transformed into a three-dimensional electron density map, into which a protein model can be fitted. The structure is refined to include solvent molecules and any additional compounds present when the protein was crystallized. As a final step, the resulting model will be submitted to the Protein Data Bank prior to publication.

NCI Cancer Center badge